Antiviral supplement formulations

ABSTRACT

The disclosure provides an oral antiviral supplement composition comprising a lysine, an ascorbic compound, a flavonoid glycoside, a threonine, and a pyridoxine. The disclosure also provides a method of reducing viral replication in a cell comprising treating a virus-infected cell with a composition of the disclosure. The disclosure further provides a method for the treatment and prophylaxis of a viral infection in a patient comprising administering a composition of the disclosure.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/609,120, filed Oct. 30, 2009, now U.S. Pat. No.9,034,834, issued May 19, 2015, which claims the benefit of U.S.Provisional Application 61/111,234; filed Nov. 4, 2008, each of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

The disclosure provides oral antiviral supplement compositionscomprising a lysine, an ascorbic compound, a flavonoid glycoside, athreonine and a pyridoxine.

2. Description of the Related Art

Influenza (the flu) is a contagious respiratory illness caused byinfluenza viruses. It can cause mild to severe illness, and at times canlead to death. According to the CDC, various subtypes of the influenzavirus circulate worldwide. It is estimated there are about 306 knownhuman influenza types, subtypes and strains and about 62 known non-polioenteroviruses which cause flu-like illness in humans. Types A and Binfluenza virus are responsible for seasonal flu epidemics each yearusually striking in December through April. Non-polio enteroviruses aresecond only to the “common cold” viruses as the most common viralinfectious agents in humans. Enteroviruses are most likely to occur inJune through November. To reduce the likelihood of contracting the A orB influenza virus, one can get a flu vaccine to provide a measure ofprotection during flu season.

The best way to prevent the flu is by getting a flu vaccination eachyear. However, the vaccination works only for prophylaxis of certainstrains of influenza. In fact, one key disadvantage of vaccines is thatthey are strain specific. Once the patient is infected, other optionsmust be considered.

In the United States there are two classes of drugs approved by the FDAfor treating or preventing influenza virus infection: the M2 ion channelblockers and the neuraminidase inhibitors (NAIs). The M2 channelblockers (adamantanes) such as amantadine and rimantadine are effectiveagainst influenza A viruses, but not influenza B viruses since they lackthe M2 protein. Use of the M2 blockers is associated with emergence ofdrug resistant mutations of the M2 protein in human influenza A virusesof H3N2 and H1N1 subtypes, and resistance has been detected in A/H5N1viruses. Therefore, the CDC has not recommended their use since 2005 andnow recommends the exclusive use of NAIs; except in possible cases ofNAI resistant strains. Viral neuraminidase may facilitate access ofvirus to cell surfaces and aid the release of newly formed virusparticles from infected cells, to allow viral infection of other cells.

If a patient contracts an A or B influenza virus strain, NAIs such asRelenza®(zanamivir) and Tamiflu®(oseltamivir) are available only byprescription and are meant to be started during the first 48 hours afteronset of symptoms. These NAI drugs have been shown to reduce theduration of a typical seven day illness by about one day. For treatmentof 2009 H1N1 virus infection, the CDC currently recommends use of eitheroseltamivir (Tamiflu®) or zanamivir (Relenza®)(http://www.cdc.gov/hlnlflu/recommendations.htm). After symptoms appearboth influenza and non-polio enteroviruses are also treatedsymptomatically to ease discomfort.

Tamiflu® (oseltamivir phosphate, Roche) is an antiviral prescriptiondrug in the form of oral capsules or oral suspensions approved for thetreatment and prophylaxis of influenza. Oseltamivir phosphate is anethyl ester prodrug requiring ester hydrolysis for conversion to theactive form, oseltamivir carboxylate. Oseltamivir carboxylate is aninhibitor of influenza virus neuraminidase affecting release of virusparticles. Influenza A virus isolates with reduced susceptibility tooseltamivir carboxylate have been recovered by serial passage of virusin cell culture in the presence of increasing concentrations ofoseltamivir carboxylate. In clinical studies of the treatment ofnaturally acquired infection with influenza virus, 1.3% ofpost-treatment isolates in adults and adolescents and 8.6% ofpost-treatment isolates in children aged 1 to 12 years showed emergenceof influenza variants with decreased neuraminidase susceptibility incell culture to oseltamivir carboxylate. Cross-resistance betweencertain zanamivir-resistant influenza mutants and oseltamivir-resistantinfluenza mutants has been observed in cell culture. During the2007-2008 influenza season, oseltamivir resistance among influenza A(H1N1) viruses increased significantly for the first time worldwide(Dharan et al., Infections with Oseltamivir-resistant influenza A(H1N1)virus in the United States, JAMA 301 (10), 1034-1041 (2009)). InfluenzaB viruses with reduced sensitivity to neuraminidase inhibitors do notarise as frequently as resistant influenza A viruses. However, theyappear to be transmitted within communities and families (Hatakeyama etal., Emergence of influenza B viruses with reduced sensitivity toneuraminidase inhibitors. JAMA, 297:13:1492-1493 (2007)).

Side effects can also occur with treatment with NAIs. For example, Rocheand the FDA informed healthcare professionals in March 2008 ofneuropsychiatric events associated with the use of Tamiflu®, in patientswith influenza. The label was revised to state that influenza can beassociated with a variety of neurologic and behavioral symptoms whichcan include events such as hallucinations, delirium, and abnormalbehavior, in some cases resulting in fatal outcomes. These events werereported primarily among pediatric patients and often had an abruptonset and rapid resolution. Because these events were reportedvoluntarily during clinical practice, estimates of frequency cannot bemade but they appear to be uncommon based on Tamiflu® usage data.Nevertheless, due to emerging drug resistant influenza strains andpossible side effects of Tamiflu, alternative antiviral therapy is ofinterest.

Relenza® (zanamivir, GlaxoSmithKline) is an antiviral prescription drugin the form of an inhalation powder approved for the treatment andprophylaxis of influenza. Zanamivir acts as an inhibitor of theinfluenza virus surface enzyme neuraminidase. Viral neuraminidase mayfacilitate access of virus to cell surfaces and aid the release of newlyformed virus particles from infected cells, to allow viral infection ofother cells. Influenza viruses with reduced susceptibility to zanamivirhave been recovered in vitro by passage of the virus in the presence ofincreasing concentrations of the drug. Zanamivir resistant influenzaviruses with a novel neuraminidase mutation have been recentlyidentified (Hurt et al., J. Virology, 83(20):10366-10373 (October2009)).

There remains great uncertainty about the usefulness of these drugs in apandemic because access may be limited and drug resistance may develop.Clearly, alternative methods of treatment and prophylaxis of viralinfection are desired to help limit development of drug-resistance toknown antiviral pharmaceuticals.

Several antiviral supplement formulations comprising various vitaminsand minerals are known and some are available commercially. Many ofthese formulations are made up of dietary supplements as defined underthe Federal Food, Drug and Cosmetic Act, Chapter II Section 201, [21U.S.C. §321], paragraph ff, and thus may be classified as dietarysupplements rather than drugs.

U.S. Pat. No. 5,626,883, Paul, discloses ascorbic acid compositionsproviding enhanced human immune system activity. The Paul compositionsinclude a water soluble ascorbate, a fat soluble ascorbyl ester, and atleast one ascorbic acid metabolite selected from basic amino acids,metabolic by-products of ascorbic acid breakdown, flavonoids, sulfurcontaining amino acids, tetrasodium pyrophosphate, and glutathione.

WO 2007/106675, Rath et al. and U.S. Pat. No. 2007/0212426, Rath et al.each disclose a composition and method of retarding viral activity andreducing viral replication comprising administering a compositioncomprising polyphenols, an ascorbic compound, lysine and proline.

U.S. Pat. No. 7,041,699, Netke et al. disclose nutrient pharmaceuticalformulations comprising polyphenols and use in treatment of cancer.Netke et al. disclose formulations comprising an ascorbic compound, theamino acids lysine, proline and N-acetylcysteine, and at least onepolyphenol.

WO 92/15315, Wilkinson, discloses a method for treating a herpesinfection which comprises administering a composition comprising lysine,vitamin C and hesperidin.

U.S. Pat. No. 5,650,418, Rath and Pauling, disclose a pharmaceuticalcomposition consisting essentially of an ascorbate compound and a lysinein the presence of a pharmaceutical carrier. Rath and Pauling alsodisclose a method of treating cardiovascular disease by administering acomposition comprising an ascorbate, nicotinic acid, a lysine, and apharmaceutical carrier.

A novel antiviral formulation comprising a lysine, an ascorbic compound,a flavonoid glycoside, a threonine and a pyridoxine has been found to besurprisingly effective at inhibition of influenza A and neuraminidaseexpression in vitro when compared to other dietary supplementformulations as well as oseltamivir.

BRIEF SUMMARY OF THE DISCLOSURE

In one embodiment, the disclosure provides an oral antiviral supplementcomposition comprising a lysine, an ascorbic compound, a flavonoidglycoside, a threonine, and a pyridoxine. In one aspect, the lysine isselected from L-lysine, L-lysine monohydrochloride, L-lysinedihydrochioride, L-lysine succinate, L-lysine glutamate, and L-lysineorotate. In another aspect, the ascorbic compound is selected from oneor more of ascorbic acid, calcium ascorbate, magnesium ascorbate,potassium ascorbate, sodium ascorbate, manganese ascorbate, zincascorbate, iron ascorbate, copper ascorbate, boron ascorbate, molybdenumascorbate, chromium ascorbate, ascorbyl palmitate, ascorbylarachidonate, ascorbyl stearate, ascorbyl linoleate, ascorbyllinoleneate, and ascorbyl oleate. In a further aspect, the flavonoidglycoside is selected from one or more of hesperidin, rutin, naringin,and quercitrin. In one specific aspect, the flavonoid glycoside is abouta 1:1 mixture by weight of hesperidin and rutin. In one aspect, thepyridoxine is pyridoxine hydrochloride. In another aspect, thecomposition further comprises taurine.

In one aspect, the composition is in a powder, capsule, lozenge, troche,tablet, liquid, or caplet form. In one aspect, the composition comprisesL-lysine monohydrochloride, ascorbic acid, hesperidin, rutin, pyridoxinehydrochloride, threonine, and taurine. In one aspect, the compositionfurther comprises calcium ascorbate, niacinamide ascorbate, and ascorbylpalmitate.

In one specific aspect, a single dose of the composition comprises fromabout 2 g to about 3.5 g L-lysine monohydrochloride; from about 0.1 g toabout 1.5 g ascorbic acid; from about 0.2 g to about 0.8 g hesperidin;from about 0.1 g to about 0.5 g rutin; from about 0.04 g to about 0.08 gpyridoxine hydrochloride; from about 0.01 g to about 0.08 g threonine;and from about 0.02 g to about 0.4 g taurine. In another specificaspect, the composition further comprises from about 0.5 g to about 0.75g calcium ascorbate; from about 0.1 g to about 0.5 g niacinamideascorbate; and from about 0.01 g to about 0.1 g ascorbyl palmitate. Inanother specific aspect, a single dose of the composition comprisesabout 3 g L-lysine monohydrochloride; from about 0.2 g to about 1.0 gascorbic acid; about 0.3 g hesperidin; about 0.3 g rutin; about 0.05 gpyridoxine hydrochloride; about 0.05 g threonine; and from about 0.02 gto about 0.3 g taurine. In a further specific aspect, a single dose ofthe composition further comprises about 0.6 g calcium ascorbate; 0.3 gniacinamide ascorbate, and about 0.05 g ascorbyl palmitate.

In another embodiment, the disclosure provides a method of reducingviral replication in a cell comprising treating a virus-infected cellwith an effective amount of a composition comprising a lysine, anascorbic compound, a flavonoid glycoside, a threonine and a pyridoxine.In one aspect, the method utilizes a composition wherein the lysine isselected from L-lysine, L-lysine monohydrochloride, L-lysinedihydrochloride, L-lysine succinate, L-lysine glutamate, and L-lysineorotate. In another aspect, the method utilizes a composition whereinthe ascorbic compound is selected from one or more of ascorbic acid,calcium ascorbate, magnesium ascorbate, potassium ascorbate, sodiumascorbate, manganese ascorbate, zinc ascorbate, iron ascorbate, copperascorbate, boron ascorbate, molybdenum ascorbate, chromium ascorbate,ascorbyl palmitate, ascorbyl arachidonate, ascorbyl stearate, ascorbyllinoleate, ascorbyl linoleneate, and ascorbyl oleate. In another aspect,the method utilizes a composition wherein the flavonoid glycoside isselected from one or more of hesperidin, rutin, naringin, andquercitrin. In another aspect, the method utilizes a composition whereinthe pyridoxine is pyridoxine hydrochloride. In another aspect, themethod utilizes a composition further comprising taurine.

In another specific aspect, the disclosure provides a method of reducingviral replication in a cell comprising treating a virus-infected cellwith an effective amount of a composition comprising L-lysinemonohydrochloride, ascorbic acid, calcium ascorbate, niacinamideascorbate, ascorbyl palmitate, hesperidin, rutin, pyridoxinehydrochloride, threonine, and taurine.

In a further embodiment, the disclosure provides a method for thetreatment or prophylaxis of a viral infection in a subject in needthereof comprising administering to the subject a therapeuticallyeffective amount of a composition comprising a lysine, an ascorbiccompound, a flavonoid glycoside, a threonine, and a pyridoxine. Incertain aspects, the viral infection is an influenza A, influenza B,influenza C or a non-polio enterovirus infection. In another aspect, themethod utilizes a composition wherein the lysine is selected fromL-lysine, L-lysine monohydrochloride, L-lysine dihydrochloride, L-lysinesuccinate, L-lysine glutamate, and L-lysine orotate. In another aspect,the method utilizes a composition wherein the ascorbic compound isselected from one or more of ascorbic acid, calcium ascorbate, magnesiumascorbate, potassium ascorbate, sodium ascorbate, manganese ascorbate,zinc ascorbate, iron ascorbate, copper ascorbate, boron ascorbate,molybdenum ascorbate, chromium ascorbate, ascorbyl palmitate, ascorbylarachidonate, ascorbyl stearate, ascorbyl linoleate, ascorbyllinoleneate, and ascorbyl oleate. In another aspect, the method utilizesa composition wherein the flavonoid glycoside is selected from one ormore of hesperidin, rutin, naringin, and quercitrin. In another aspect,the method utilizes a composition wherein the pyridoxine is pyridoxinehydrochloride. In another aspect, the method utilizes a compositionfurther comprising taurine. In another aspect, the method utilizes acomposition comprising L-lysine monohydrochloride, ascorbic acid,calcium ascorbate, niacinamide ascorbate, ascorbyl palmitate,hesperidin, rutin, pyridoxine hydrochloride, threonine, and taurine. Inanother aspect, the method utilizes a composition that further comprisescalcium ascorbate, niacinamide ascorbate, and ascorbyl palmitate.

In one specific aspect, the method for the treatment or prophylaxis of aviral infection in a subject in need thereof comprises administering tothe subject a therapeutically effective amount of a composition whereina single dose comprises from about 2 g to about 3.5 g L-lysinemonohydrochloride; from about 0.1 g to about 1.5 g ascorbic acid; fromabout 0.2 g to about 0.8 g hesperidin; from about 0.1 g to about 0.5 grutin; from about 0.04 g to about 0.08 g pyridoxine hydrochloride; fromabout 0.01 g to about 0.08 g threonine; and from about 0.02 g to about0.4 g taurine. In a further specific aspect, the method utilizes acomposition wherein a single dose further comprises from about 0.5 g toabout 0.75 g calcium ascorbate; from about 0.1 g to about 0.5 gniacinamide ascorbate; and from about 0.01 g to about 0.1 g ascorbylpalmitate. In another specific aspect, the method utilizes a compositionwherein a single dose comprises about 3 g L-lysine monohydrochloride;from about 0.2 g to about 1.0 g ascorbic acid; about 0.3 g hesperidin;about 0.3 g rutin; about 0.05 g pyridoxine hydrochloride; about 0.05 gthreonine; and from about 0.02 g to about 0.3 g taurine. In a furtherspecific aspect, the method utilizes a composition wherein a single dosefurther comprises about 0.6 g calcium ascorbate; 0.3 g niacinamideascorbate, and about 0.05 g ascorbyl palmitate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a concentration (uM)/response graph for in vitro inhibitionof influenza A infection of Vero cells when treated with variousformulations in a branched chain DNA (bDNA) assay of infected cellsupernatants as described in Example 2. The data represents the mean(n=3) for each data point, but error bars have been omitted for clarity.

FIG. 2 shows a concentration (uM)/response graph for in vitro inhibitionof influenza A infection of Vero cells when treated with variousformulations in a bDNA assay as described in Example 2. The datarepresents the mean (n=3) for each data point; the error bars representstandard error of the mean (SEM).

FIG. 3 shows inhibition of neuraminidase from influenza A infected Verocell supernatants in vitro by various formulations in the Amplex RedNeuraminidase assay as described in Example 3. The data represents themean (n=3) for each data point, but error bars have been omitted forclarity.

FIG. 4 shows inhibition of neuraminidase from influenza A infected Verocell supernatants in vitro by two formulations in the Amplex RedNeuraminidase assay as described in Example 3. The data represents themean (n=3) for each data point; the error bars represent standard errorof the mean (SEM).

FIG. 5 shows inhibition of matrix metalloproteinase 9 (MMP-9) expressionin influenza-A-infected Vero cells by various formulations (uM) asdescribed in Example 4. The data represents the mean (n=3) for each datapoint, but error bars have been omitted for clarity.

FIG. 6 shows inhibition of matrix metalloproteinase 9 (MMP-9) expressionin influenza-A-infected Vero cells by two formulations as described inExample 4. The data represents the mean (n=3) for each data point; theerror bars represent standard error of the mean (SEM).

FIG. 7 shows a table (Table 4) of various compositions tested in theNeuraminidase assay.

FIG. 8 shows a graph of neuraminidase activity of human influenza virusinfected cells after exposure to oseltamivir carboxylate, Formula V andten test compositions from Table 4 containing various components ofFormula V.

FIG. 9 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine alone (1) or lysine with ascorbic acid/ascorbates (1/2; 9A).

FIG. 10 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/flavonoid glycosides (1/2/3; 10A) orlysine/ascorbates/pyridoxine (1/2/4; 10B).

FIG. 11 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/threonine (1/2/5; 11A) or lysine/ascorbates/taurine(1/2/6; 11B).

FIG. 12 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/pyridoxine HCl/threonine (1/2/4/5; 12A) orlysine/ascorbates/flavonoid glycosides/threonine (1/2/3/5; 12B).

FIG. 13 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/flavonoid glycosides/pyridoxine HCl/threonine(1/2/3/4/5; 13A) or lysine/ascorbic acid/flavonoid glycosides/pyridoxineHCl/threonine/taurine (1/2/3/4/5/6; 13B).

DETAILED DESCRIPTION

The disclosure provides novel antiviral supplement compositionscomprising a lysine, an ascorbic compound, a flavonoid glycoside, athreonine, and a pyridoxine. The disclosure provides compositions in theform of a powder, capsule, tablet, lozenge, troche, liquid, or caplet. Aspecific embodiment discloses a powder comprising lysine, ascorbic acid,flavonoid glycosides, pyridoxine hydrochloride, taurine, and threonine.

The term “patient” or “subject” as used herein refers to an animal, forexample a mammal, such as a human, who is the object of treatment. Thesubject, or patient, may be either male or female.

The term “about” as used herein refers to a numeric range that is +1-10%of the given quantity. For example, the term “about 50%” refers to 45%to 55%, and “about 100 mg” refers to 90 mg to 110 mg.

The term “virus” used herein refers to any of a large group ofsubmicroscopic agents that consist of a segment of DNA or RNA surroundedby a coat of protein. Influenza viruses and enteroviruses are RNAviruses. The virus is a parasite that needs a host cell to replicate.Because viruses are unable to replicate without a host cell, they arenot considered living organisms in conventional taxonomic systems. Theyare described as “live” when they are capable of replicating and causingdisease. Accordingly, the term “viral activity” refers to any state ofbeing active or any energetic action or movement or liveliness of avirus. Accordingly, the term “viral replication” refers to any processby which genetic materials, a single-celled organism, or a virusreproduces or makes a copy of itself.

The term “Neuraminidase” as used herein refers to a viral neuraminidaseprotein that acts as a hydrolytic enzyme that removes sialic acid frommucoproteins and is found chiefly in microorganisms of the respiratoryand intestinal tracts. Neuraminidase breaks the bonds that hold newvirus particles to the outside of an infected cell. Once the enzymebreaks these bonds, this sets free new viruses that can infect othercells and spread infection.

The term “Neuraminidase inhibitor” (NAI) as used herein refers to a drugor formulation that blocks the function of neuraminidase protein andtherefore prevents new virus particles from being released, therebylimiting the spread of infection. Specific NAIs include oseltamivir,zanamivir, laninamivir and peramivir.

The term “Vero cells” as used herein refers to the African green monkeykidney cell line which is a suitable system for the primary isolationand cultivation of influenza A viruses. It is also known that Vero cellsare suitable for isolation and productive replication of influenza Bviruses.

The term “infection” as used herein refers to the presence of a virus inor on a subject, which if replication of the virus was retarded or ofthe activity of the virus was reduced, would result in a benefit to thesubject. Accordingly, the term “infection” refers to the presence ofpathogens at any anatomical site of a human or animal.

The term “treating” as used herein refers to the administration of acompound or composition to a subject for therapeutic purposes. The term“administration” includes delivery to a subject by any appropriatemethod which serves to deliver the drug to the site of the infection.The administration of the drug can be oral, nasal, parental, topical,ophthalmic, or transdermal administration or delivery in the form ofsolid, semi-solid, lyophilized powder, or liquid dosage forms. Thedosage forms include tablets, capsules, troches, powders, solutions,suspensions, suppositories, or the like, preferably in unit dosage formssuitable for simple administration of precise dosages.

The term “antiviral supplement” as used herein includes any compositionused specifically for treatment or prophylaxis of viral infections,particularly influenza virus or non-polio enterovirus infections. Thecompositions of the disclosure can be evaluated in one aspect by variousneuraminidase assays. In another aspect, the compositions of thedisclosure can be evaluated by retarding the growth and reproduction ofviruses in a cell based assay. In another aspect, the compositions ofthe disclosure can be evaluated by decreasing the duration of a viralinfection in a patient. In another aspect, the compositions of thedisclosure can be evaluated by decreasing the severity or duration ofsymptoms of a viral infection in a patient.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which retain the biological effectiveness and properties ofthe active ingredient of the biochemical composition, which are nototherwise undesirable. Pharmaceutically acceptable salts include, butare not limited to, salts of sodium, potassium, calcium, magnesium,aluminum and the like.

As used herein, the term “an effective amount” refers to that an amountof a composition of the disclosure that when administered to anindividual subject in need thereof, is sufficient to reduce the virusactivity and/or growth thereby enhancing the antiviral activity.

As used herein, the term “therapeutically effective amount” refers to anamount of a composition of the disclosure that when administered to ahuman subject in need thereof, is sufficient to effect treatment orprophylaxis for influenza virus infection, or non-polio enterovirusinfection. The amount that is therapeutically effective will depend uponthe patient's size and gender, the stage and severity of the infectionand the result sought. For a given patient and condition, atherapeutically effective amount can be determined by methods known tothose of skill in the art. For example, in reference to the treatment ofa influenza virus infection using the compositions of the presentinvention, a therapeutically effective amount refers to that amount ofthe composition which has the effect of (1) reducing the shedding of thevirus, (2) reducing the duration of the infection, (3) reducinginfectivity and/or, (4) reducing the severity (or, preferably,eliminating) one or more other symptoms associated with the infectionsuch as, for example, fever, headache, fatigue, dry cough, sore throat,muscle aches, conjunctivitis, runny and/or stuffy nose. Such aneffective dose will generally depend on the factors described above. Aprophylactically effective dose is one that reduces the likelihood ofcontacting an influenza virus infection, or non-polio enterovirusinfection. A prophylactically effective dose is from about 20% to about100%, preferably from about 40% to about 60%, of a therapeuticallyeffective dose.

In one aspect, the administration is oral administration. Generally, atherapeutically effective dose is not less than about 10% and not morethan about 200% of the amounts of individual ingredients listed inTable 1. In certain aspects, a therapeutically effective dose fortreatment of a viral infection is from about 50% to about 150%, or fromabout 80% to about 120%, or about 100% identical with the list ofcomponents in Table 1. In another aspect, a therapeutically effectivedose for treatment of a viral infection in a subject in need thereof isadministered every 4 to 6 waking hours, or from about two to six timesper day. In a further aspect, a therapeutically effective dose forprophylaxis of a viral infection in a subject in need thereof isadministered every 8 to 12 waking hours, or from about one to threetimes per day.

The term, “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the compositions of theinvention from one organ, or portion of the body, to another organ, orportion of the body without affecting its biological effect. Eachcarrier should be “acceptable” in the sense of being compatible with theother ingredients of the composition and not injurious to the subject.

Symptoms of influenza can include fever, headache, fatigue, dry cough,sore throat, muscle aches, runny and/or stuffy nose. Gastrointestinalsymptoms such as nausea, vomiting and diarrhea can also occur, but aremore common in children than adults.

Symptoms of non-polio enteroviruses include fever, muscle aches, upperrespiratory symptoms, tiredness, and flu-like illness with rash.

Viruses spread from person to person primarily through coughing orsneezing. People may also become infected by contact with contaminatedsurfaces or objects, then by touching their mouth or nose. Most healthyadults may be able to infect others beginning about one day beforesymptoms develop and up to about five days after becoming ill.

Influenza viruses are dynamic and are continuously evolving. Influenzaviruses can change in two different ways: antigenic drift and antigenicshift. Influenza viruses are constantly changing by antigenic drift, butantigenic shift occurs only occasionally. Influenza type A virusesundergo both kinds of changes; influenza type B viruses change only bythe more gradual process of antigenic drift. Antigenic drift refers tosmall, gradual changes that occur through point mutations in the twogenes that contain the genetic material to produce the main surfaceproteins, hemagglutinin, and neuraminidase. These point mutations occurunpredictably and result in minor changes to these surface proteins.Antigenic drift produces new virus strains that may not be recognized byantibodies to earlier influenza strains.

Humans can be infected with influenza types A, B, and C viruses.Influenza A, B, and C are responsible for producing 306 human influenzaviruses. Subtypes of influenza A that are currently circulating amongpeople worldwide include H1N1, H1N2, and H3N2 viruses.

Based on global experience to date, 2009 H1N1 influenza viruses likelywill be the most common influenza viruses among those circulating in thecoming flu season, particularly those causing influenza among youngerage groups. (CDC “Updated Interim Recommendations for the Use ofAntiviral Medications in the Treatment and Prevention of Influenza forthe 2009-2010 Season, Sep. 22, 2009,http://www.cdc.gov/hlnlflu/recommendations.htm)

Wild birds are a natural host for known subtypes of influenza A viruses.Typically, wild birds do not become sick when they are infected withavian influenza A viruses. However, domestic poultry can become verysick and die from avian influenza.

Influenza type A viruses are divided into subtypes and named on thebasis of two proteins on the surface of the virus: hemagglutinin (HA)and neuraminidase (NA). There are 16 known HA subtypes and 9 known NAsubtypes. Many different combinations of HA and NA proteins arepossible. Only some influenza A subtypes (i.e., H1N1 “Spanish Flu” or“Swine Flu”, H1N2, and H3N2 “Hong Kong Flu”) are currently in generalcirculation among people. Only influenza A viruses infect birds, and allknown subtypes of influenza A viruses can infect birds. However, thereare substantial genetic differences between the influenza A subtypesthat typically infect birds and those that infect both people and birds.Three prominent subtypes of the avian influenza A viruses are known toinfect both birds and people.

Influenza A H7 includes several subtypes. H7 infection in humans is rarebut can occur among persons who have direct contact with infected birds.Symptoms may include conjunctivitis and/or upper respiratory symptoms.H7 viruses have been associated with both LPAI (e.g., H7N2, H7N7) andHPAI (e.g., H7N3, H7N7), and have caused mild to severe and fatalillness in humans.

Influenza A H5 includes several known subtypes including the highlypathogenic H5N1. A bird adapted strain of H5N1 is called HPAI H5N1(highly pathogenic avian influenza virus of type A of subtype H5N1),commonly known as “avian influenza” or “bird flu”. HPAI H5N1 viruses arecurrently circulating in Asia and Europe and can cause severe illness ordeath. HPAI H5N1 is considered a potential agricultural bioweapon. Lowpathogenic avian influenza H5N1 (LPAI H5N1), also called “NorthAmerican” H5N1, occurs in wild birds and causes minor illness or nonoticeable signs in birds and is not known to affect humans, but it ispossible to be transmitted to poultry and possibly mutate to a highlypathogenic strain.

Low pathogenic avian influenza A (H9N2) was confirmed in 1999. Severalpotential subtypes of H9 are known; however, influenza A H9 has rarelybeen reported to infect humans. This subtype has been documented only ina low pathogenic form.

Influenza B viruses are usually found only in humans. Unlike influenza Aviruses, these viruses are not classified according to subtype.Influenza B viruses can cause morbidity and mortality among humans, butin general are associated with less severe epidemics than influenza Aviruses. Although influenza type B viruses can cause human epidemics,they have not caused pandemics.

Influenza type C viruses cause mild illness in humans and do not causeepidemics or pandemics. These viruses are not classified according tosubtype.

Influenza B viruses and subtypes of influenza A virus are furthercharacterized into strains. There are many different strains ofinfluenza B viruses and of influenza A subtypes. New strains ofinfluenza viruses appear and replace older strains. This process occursthrough antigenic drift. When a new strain of human influenza virusemerges, antibody protection that may have been developed afterinfection or vaccination with an older strain may not provide protectionagainst the new strain.

Evidence that influenza can be more severe in pregnant women isavailable from observations during previous pandemics and from studiesamong pregnant women who had seasonal influenza. An excess ofinfluenza-associated deaths among pregnant women were reported duringthe pandemics of 1918-1919 and 1957-1958. Adverse pregnancy outcomeshave been reported following previous influenza pandemics, withincreased rates of spontaneous abortion and preterm birth reported,especially among women with pneumonia(http://www.cdc.gov/H1N1flu/clinicianpregnant.htm, Jun. 30, 2009).Pregnant women and women up to 2 weeks postpartum are considered to beat higher risk of complications from influenza infection.

Oseltamivir, zanamivir, amantidine and rimantidine are “PregnancyCategory C” medications indicating no studies have been performed toassess the safety of these drugs in pregnant women. Limited cases ofamantadine use for severe influenza illness during the third trimesterhave been reported. However, both amantadine and rimantadine have beendemonstrated in animal studies to be teratogenic and embryotoxic whenadministered at substantially high doses. Because of the unknown effectsof influenza antiviral drugs on pregnant women and their fetuses, thesefour drugs should be used during pregnancy only if the potential benefitjustifies the potential risk to the embryo or fetus (Prevention &Control of Influenza—Recommendations of the Advisory Committee onImmunization Practices (ACIP) 2004. MMWR 2004 May 28; 53(RR06);1-40.)None-the-less, the CDC states that the available risk-benefit dataindicate that pregnant women with suspected or confirmed influenzashould receive prompt antiviral treatment.

Matrix metalloproteinases (MMPs) are protein enzymes involved in thebreakdown of extracellular matrix in normal physiological processes suchas tissue remodeling, reproduction and embryonic development. MMPs areimportant enzymes in tissue remodeling, a key event for the developmentof fetal membranes and placenta and establishing the feto-maternalinterface during early pregnancy. Most MMPs are secreted as inactiveproteins which are activated when cleaved by extracellular proteinases.MMP-2 (72 kDa type IV collagenase) and MMP-9 (gelatinase B, 92kD type IVcollagenase) are key effectors of extracellular matrix remodeling.Studies indicate MMP-2 and MMP-9 are present in first trimesterextra-embryonic coelomic fluid and in the amniotic fluid at allgestations. The predominant MMP activated protein is the latent form ofMMP-2 which is found present in increasing concentrations in amnioticfluid from the first to the second trimester (Riley et al., Secretion ofmatrix metalloproteinase-2, matrix metalloproteinase-9 and tissueinhibitor of metalloproteinases into the intrauterine compartmentsduring early pregnancy. Molecul. Human Reprod. 5(4): 676-381 (1999)).Tamiflu® inhibits expression of MMP-9, as shown in FIG. 5, which maycontribute to increased risk in pregnancy.

In one embodiment, compositions of the disclosure only weakly inhibitthe expression of MMP-9, which may result in greater safety in thetreatment of influenza in pregnant women. Data is shown in FIGS. 5 and6. In another embodiment, compositions of the disclosure have beendemonstrated to show no significant inhibition of expression of MMP-2,as discussed in Example 5, which may result in greater safety in thetreatment of influenza in pregnant women.

Enteroviruses are small viruses that are made of ribonucleic acid (RNA)and protein. This group includes the polioviruses, coxsackieviruses,echoviruses, and other enteroviruses. There are 62 non-polioenteroviruses that can cause disease in humans: 23 Coxsackie A viruses,6 Coxsackie B viruses and 28 echoviruses and 5 other enteroviruses.

Non-polio enteroviruses are very common and second only to the “commoncold” virus, called the rhinovirus, as the most common viral infectiousagent in humans. Everyone without immunity to a specific enterovirus isat risk of infection. Enterovirus outbreaks commonly occur during thesummer through fall.

The present disclosure was developed based upon the recognition that acombination of the supplements L-lysine and ascorbic acid is somewhateffective in reducing viral replication and reducing viral infection. Inone embodiment, the disclosure provides an antiviral supplementcomposition which surprisingly further reduces viral replication andinhibits viral infection. Specifically, the disclosure provides anantiviral supplement composition comprising a lysine, an ascorbiccompound, a flavonoid glycoside, a pyridoxine, taurine and threoninewhich has been found to have significantly improved antiviralproperties.

In another embodiment, the disclosure provides a method of reducing theseverity and duration of symptoms of an influenza infection in a humanby administering the composition. In this embodiment, the compositionsare taken at the first signs of flu-like illness. In one aspect, thecomposition is administered every six waking hours with water untilsymptoms are resolved. In another aspect, the compositions areadministered about every four hours after first symptoms appear. Inanother aspect, the composition is administered twice a day untilsymptoms are resolved.

In another embodiment, the composition of the present disclosure reducesinfluenza viral infection of cells. In one aspect, the compositions actas neuraminidase inhibitors. In another aspect, compositions of thepresent disclosure weakly inhibit MMP-9 expression in vitro.

It is known that vitamin C (ascorbic acid) is necessary for general goodhealth in humans. Humans lack the biochemical mechanisms to synthesizevitamin C, so it must be supplied in the diet in the form of food orsupplement. Englard, S. and Steifter, S. “The Biochemical functions ofAscorbic Acid,” Ann. Rev. Nutr. 1986. Except where inborn errors ofmetabolism such as cystinuria, oxalosis, and hyperuricemia are present,it is known that vitamin C can be taken in megadoses without toxic sideeffects. See Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S.,1972, The metabolic basis of inherited disease. 3rd ed. McGraw Hill.

Vitamin C supports the function of the human immune system. Vitamin C isthought to stimulate the human immune system by enhancing interferonsynthesis and lymphocyte activity, particularly the class of lymphocytereferred to as natural killer (NK) cells. Sigel, B. V. & Morton, J. I.“Vitamin C and Immunity: Natural Killer (NK) cell factor” Int. J.Vitamin & Nutrition Res. 1983, 53:179-183; Lovzova, E., Savary, C. A., &Heberman, R. B. “Induction of NK cells activity against fresh humanleukemia in culture with interlukin 2” J. Immunology 1987,138:2718-2727. Natural killer cells are lymphocytes that spontaneouslykill tumor or virus-infected cells. Decreased numbers of circulating NKcells have been linked to the development and progression of variousimmunodeficiencies, viral infections, AIDS, and cancer. Vitamin C isalso believed to provide further benefits by acting as an antioxidantaiding the immune system by reducing the amount of free radical damagethat can occur as a result of normal body metabolism as well as fromexogenous sources.

Studies have shown that vitamin C supplementation (e.g., 500 mg/day)increases plasma concentrations of glutathione. Johnston et al. “VitaminC Elevates Red Blood Cell Glutathione in Healthy Adults” Am. J. Clin.Nutr. 1993, 58:103-105. Certain data suggest that the thiol antioxidantglutathione (GSH) has an anti-influenza activity in vitro and in vivo.Oxidative stress or other conditions that deplete GSH in the epitheliumof the oral, nasal, and upper airway may, therefore, enhancesusceptibility to influenza infection.

It is known that infection by RNA virus induces oxidative stress in hostcells. Accumulating evidence suggests that cellular redox status playsan important role in regulating viral replication and infectivity.Certain data suggest that the thiol antioxidant GSH has ananti-influenza activity in vitro and in vivo. Specifically, experimentswere performed to determine whether the thiol antioxidant glutathione(GSH) blocked influenza viral infection in cultures of Madin-Darbycanine kidney cells or human small airway epithelial cells. Protectionagainst production of active virus particles was observed at a low(0.05-0.1) multiplicity of infection (MOD. Cai et al. 2003, Inhibitionof influenza infection by glutathione. Free Rad. Biol. And Med. 34 (7):928-936. It was also found that GSH inhibited expression of viral matrixprotein and inhibited virally induced caspase activation and Fasupregulation. Together, the data suggest that the thiol antioxidantglutathione (GSH) has an anti-influenza activity in vitro and in vivo.Oxidative stress or other conditions that deplete GSH in the epitheliumof the oral, nasal, and upper airway may, therefore, enhancesusceptibility to influenza infection.

In one embodiment, the compositions of the present disclosure comprisevitamin C as one or more forms of ascorbic acid, ascorbyl ester orascorbate salt; together termed ascorbic compounds. In one aspect, theone or more ascorbic compounds are selected from any biologicallyacceptable form of an ascorbic acid, ascorbyl ester or ascorbateincluding either or both water-soluble and fat-soluble forms. The watersoluble form of ascorbic acid can be selected from the group consistingof ascorbic acid, a biologically acceptable mono or divalent metal ionsalt of ascorbic acid and niacinamide ascorbate, and mixtures thereof.Suitable metal ion salts of ascorbic acid are those selected from thegroup consisting of calcium ascorbate; magnesium ascorbate; potassiumascorbate; and sodium ascorbate, either alone or some mixture thereof.Other water soluble forms can include manganese ascorbate; zincascorbate; iron ascorbate; copper ascorbate; boron ascorbate; molybdenumascorbate; and chromium ascorbate. The fat soluble ascorbyl esterspreferably comprise fatty acid esters of saturated or unsaturatedcarboxylic acids with ascorbyl palmitate being one preferred form. Otherfat soluble esters of ascorbic acid which are preferred include:ascorbyl palmitate; ascorbyl arachidonate; ascorbyl stearate; ascorbyllinoleate; ascorbyl linoleneate; and ascorbyl oleate.

In one aspect, the composition comprises from about 15% to about 35% byweight, preferably about 20% to about 30% by weight combined of one ormore ascorbic compounds, or equivalent. In a specific aspect, thecomposition comprises about 25% by weight of combined ascorbic acid,calcium ascorbate, niacinamide ascorbate, and ascorbyl palmitate. Inanother aspect, the composition comprises about 0.5 to about 2.0 g,preferably about 0.5 g to about 1.5 g combined weight of one or moreascorbic compounds per dose. In one aspect, certain compositionscomprise m about 0.1 g to about 1.5 g ascorbic acid per dose. In anotheraspect, certain compositions of the disclosure comprise from about 0.5 gto about 0.75 g calcium ascorbate per dose. In a further aspect, certaincompositions of the disclosure comprise from about 0.1 g to about 0.5 gniacinamide ascorbate per dose. In another aspect, certain compositionsof the disclosure comprise from about 0.01 g to about 0.1 g ascorbylpalmitate per dose.

Lysine is one of the essential amino acids found in proteins. It isneeded for proper growth in infants and for maintenance of nitrogenbalance in adults. L-lysine is metabolized in mammals to give acetyl-CoAby an initial transamination with alpha-ketoglutarate. Lysine isessential for protein synthesis in the body. L-lysine plays a role incalcium absorption, building muscle protein, recovery from injury, andthe body's production of hormones, enzymes and antibodies. L-lysine isknown to be used to treat mouth and genital lesions caused by herpessimplex virus as well as shingles caused by herpes zoster viruses.Taking lysine supplements can speed recovery time and reduce the chanceof recurrent breakouts of the herpes virus infection. General lysinedoses for the treatment of symptoms of herpes virus infection includeoral divided doses of about 3 g to about 9 g per day. Lysine supplementsare considered generally safe and non-toxic. About 0.5 g to about to 1.5g per day lysine is suggested to prevent recurrences of outbreak.Griffith et al. Success of L-lysine therapy in frequently recurrentherpes simplex infection. Treatment and prophylaxis. Dermatologica.1987; 175(4):183-190. Proteins of the herpes simplex virus are rich inL-arginine and tissue culture studies have shown that when the ratio ofL-lysine to L-arginine is high, viral replication and cytopathogenicityof the herpes simplex virus have been found to be inhibited.

In one embodiment, the compositions of the present disclosure compriselysine. The term lysine refers to any pharmaceutically acceptable formincluding a salt of L-lysine which includes lysine hydrochloride, lysinedihydrochloride, lysine succinate, lysine glutamate, lysine orotate aswell as L-lysine. Other acceptable forms of lysine include lysinederivatives such as lysine acetate. In one aspect, the compositioncomprises from about 40% to about 80% by weight, preferably about 50% toabout 70% by weight of L-lysine hydrochloride, or equivalent. In aspecific aspect, the composition comprises about 60% by weight ofL-lysine hydrochloride. In one aspect, the compositions of thedisclosure comprise a single dose range of from about 1,000 mg to about5,000 mg, preferably about 2,000 to about 3,500 mg.

Flavonoids are a large family of compounds synthesized by plants thathave a common chemical structure. Flavonoids are further divided intosubclasses based upon chemical structure. Flavonoids are found in fruitsand vegetables in the diet. Flavonoids connected to one or more sugarmolecules are known as flavonoid glycosides, while those that are notconnected to a sugar molecule are called aglycones. With the exceptionof flavanols (catechins and proanthocyanidins), flavonoids occur inplants and most foods as glycosides. Even after cooking, most flavonoidglycosides reach the small intestine intact. Only flavonoid aglyconesand flavonoid glucosides (bound to glucose) are absorbed in the smallintestine, where they are rapidly metabolized to form methylated,glucuronidated, or sulfated metabolites. The ability of flavonoids tochelate (bind) metal ions appears to contribute to their antioxidantactivity in vitro. Although it was initially hypothesized that thebiological effects of flavonoids would be related to their antioxidantactivity, available evidence from cell culture experiments suggests thatmany of the biological effects of flavonoids are related to theirability to modulate cell-signaling pathways (Williams et al.,Flavonoids: antioxidants or signalling molecules? Free Radic. Biol.Med.; 36(7):838-849 (2004)).

In one aspect, the compositions of the present disclosure comprise oneor more flavonoid glycosides. The flavonoid glycosides may include anyflavonoid which is glycosylated. In a specific aspect, the compositionscomprise one or more flavonoid glycosides selected from hesperidin,rutin, naringin, and quercitrin. In one specific aspect, thecompositions comprise hesperidin and rutin.

In one aspect, the composition comprises from about 5% to about 20% byweight, preferably about 10% to about 15% by weight combined of one ormore flavonoid glycoside, or equivalent. In a specific aspect, thecomposition comprises about 12% by weight of combined hesperidin complexand/or rutin. In one aspect, the compositions comprise about from about0.2 g to about 0.8 g hesperidin per single dose. In another aspect, thecompositions comprise from about 0.1 to about 0.5 g rutin per dose.

Threonine is an essential amino acid that is a necessary building blockfor protein. It promotes the growth of the thymus, a small gland thatregulates many of the hormones and cells vital to immune defense. Even amoderate reduction in dietary intake of threonine can produce adepression in immune response or antibody production. Lotan. Humoral andcellular immune response in growing rats fed a 10% gluten diet. Isr. J.Med. Sci. 1989 August; 25(8):437-41. Recent research suggests that theeffects of threonine relate to a specific requirement by the thymus forthis amino acid and its ability to promote cell immune defensefunctions. Braverman, Threonine: The Immunity Booster, The HealingNutrients Within, 2003; 13 pp: 201. In one embodiment, the compositionsof the present disclosure comprise threonine. The threonine can beselected from L-threonine or any pharmaceutically acceptable salt orderivative.

In a specific aspect, the threonine is L-threonine. In one aspect, thecomposition comprises from about 0.1% to about 5% by weight, preferablyabout 0.5% to about 2% by weight of L-threonine, or equivalent. In aspecific aspect, the composition comprises about 1% by weight ofL-threonine. In one aspect, the compositions of the disclosure comprisefrom about 0.01 to about 0.08 g threonine per dose.

In one embodiment, the compositions of the present disclosure comprisepyridoxine. Pyridoxine is one form of vitamin B6. Pyridoxine is utilizedby the liver to synthesize pyridoxal phosphate (PLP), the activecoenzyme form. PLP is a cofactor for the enzyme threonine aldolase whichcatalyzes the conversion of hydroxy-N-trimethyl-L-lysine totrimethylaminobutyraldehyde; intermediates in the conversion of L-lysineto L-carnitine. Pyridoxine deficiency is known to produce a significantreduction in plasma carnitine levels. Absorption and Utilization ofAmino Acids, Vol. II, Mendel Friedman, CRC Press, 1989, ISBN 0849360072,Ch. III, p. 48-49. Pyridoxine is a water soluble B vitamin which servesas a cofactor and is involved in the metabolism of protein,carbohydrates, and the production of insulin and red and white bloodcells. Vitamin B6 is essential in numerous biochemical pathways in theimmune system. Pyridoxine deficiency leads to impairment of immuneresponses (Trakatellis et al., 1997, Pyridoxine deficiency: newapproaches in immunosuppression and chemotherapy. Postgrad. Med. J.October; 73(864): 617-622 (1997)). Vitamin B6 is usually safe at intakesup to 200 mg per day in adults.

In one embodiment, the compositions of the present disclosure comprisepyridoxine. In one specific aspect, the pyridoxine is pyridoxinehydrochloride. In one aspect, the composition comprises from about 0.1%to about 2% by weight, preferably about 0.5% to about 1.5% by weight ofpyridoxine hydrochloride, or equivalent. In a specific aspect, thecomposition comprises about 1% by weight of pyridoxine hydrochloride. Inone aspect, compositions of the disclosure comprise from about 0.04 g toabout 0.08 g pyridoxine hydrochloride per dose.

In one embodiment, the compositions of the present disclosure comprisetaurine. Taurine, or 2-aminoethanesulfonic acid, is a metabolite of thesulfur-containing amino acid, cysteine. Taurine is one of the few knownnaturally occurring sulfonic acids. Metabolic actions of taurine includebile acid conjugation, detoxification, membrane stabilization,osmoregulation, and modulation of cellular calcium levels. Taurine isable to cross the blood brain barrier. Taurine acts as an antioxidantand protects against toxicity of various substances (Green et al.,Antioxidant role and subcellular location of hypotaurine and taurine inhuman neutrophils. Biochimica et biophysica acta Jan 23; 1073(1):91-7(1991)). Taurine is also known to play a role in the immune system. Forexample, taurine interacts with hypochlorous acid, produced during the“oxidant burst” of stimulated macrophages, to produce taurine chloramine(TauCl). This compound may have important immunomodulatory propertiesand may be responsible for properties that have been ascribed earlier totaurine. In vitro studies have shown that an increase in taurineconcentration from physiological to superphysiological concentrationshas no effect on proinflammatory cytokine production by peripheral bloodmononuclear cells; however, Tau-Cl modulates synthesis ofpro-inflammatory cytokines, and therefore it may play a role in theinitiation and propagation of immune response (Chorazy et al., Taurinechloramine modulates cytokine production by human peripheral bloodmononuclear cells. Amino Acids. 23:407-13 (2002)). Tau-Cl inhibitsnuclear factor κB activation and the capacity for proinflammatorycytokine production, producing an anti-inflammatory effect (Huxtable RJ., Taurine past, present, and future. Adv Exp Med Biol. 403:641-50(1996)). Although the maximum safe level of dietary taurine has not beenestablished, 0.9 to 1.4 grams per day have been tolerated withoutdocumented adverse effects (Braverman, Taurine: The Seizure Fighter, TheHealing Nutrients Within, Basic Health Publications, Inc., Laguna Beach,Calif., Ch. 8, pp: 132-133 (2003)).

In one embodiment, compositions of the present disclosure optionallycomprise a taurine, or pharmacologically acceptable salt or derivativethereof. In one aspect, the composition comprises from about 1% to about10% by weight, preferably about 2% to about 6% by weight of taurine, orequivalent. In a specific aspect, the composition comprises about 4% byweight of taurine. In one aspect, compositions of the disclosurecomprise from about from about 0.02 g to about 0.4 g taurine per dose.

The compositions of the present disclosure are oral compositions. In oneembodiment, the oral composition can be in the form of a powder,capsule, tablet, troche, liquid, or caplet. The powder may be utilizedin a capsule fill, or sold in a single dose packet meant to mix with afood such as applesauce, or can be an effervescent powder formulationsold in a single dose packet and meant for suspension in a liquid. Inone aspect, the capsule, tablet, lozenge is intended for ingestion byswallowing. In another aspect, the tablet, capsule, or lozenge is orallydisintegrable. In one aspect, the tablet, capsule, lozenge or troche isa slow release composition. In another aspect, the tablet, lozenge,troche or capsule is an immediate release composition. In anotheraspect, the oral composition can be a prepackaged liquid drink, whereinthe formulation is suspended in a flavored liquid. In preferred aspects,the composition is in the form of a tablet, a capsule, or a powder meantto mix with a food, such as applesauce. Although the compositions of thedisclosure are primarily oral forms, other modes of administration suchas parenteral forms, or anal suppositories have been contemplated.

The tablet, capsule, and caplet forms of the disclosure may comprise,aside from those components specified above, other various additives,such as vehicle, binder, disintegrating agent, lubricant, thickener,surfactant, osmotic pressure regulator, electrolyte, sweetener,flavoring, perfume, pigment, pH regulator and others appropriately asrequired.

Specifically, the additives include starches such as wheat starch,potato starch, corn starch, and dextrin, sugars such as sucrose,glucose, fructose, maltose, xylose, and lactose, sugar alcohols such assorbitol, mannitol, maltitol, and xylitol, isotransposable glycosidessuch as coupling sugar and paratinose, vehicles such as calciumphosphate and calcium sulfate, binders and thickeners such as starch,sugar, gelatin, gum arabic, dextrin, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, hydroxy propyl cellulose, xanthan gum,pectin, tragacanth gum, casein, and alginic acid, lubricants such asleucine, isoleucine, valine, sugar-ester, hardening oil, stearic acid,magnesium stearate, talc, and macrogol, disintegrating agents such asavicel, CMC, CMC-Na and CMC-Ca, surfactants such as polysorbate andlecithin, and sweeteners such as sugars, sugar alcohols, aspartame,alitame, other dipeptides, stevia, and saccharin, and they may be usedin proper amounts selectively in consideration of the relation with theessential components, property of the composition, manufacturing method,etc.

In another embodiment, compositions of the disclosure can optionallyfurther comprise one or more flavoring agents. The optional flavoringagent is added to increase patient acceptability and compliance with therecommended dosing schedule. The flavoring agents that may be usedinclude those flavors known to the skilled artisan, such as natural andartificial flavors. These flavorings may be chosen from synthetic flavoroils and flavoring aromatics and/or oils, oleoresins and extractsderived from plants, leaves, flowers, fruits, and so forth, andcombinations thereof. Non-limiting representative flavor oils includespearmint oil, cinnamon oil, oil of wintergreen (methyl salicylate),peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, thymeoil, cedar leaf oil, oil of nutmeg, allspice, oil of sage, mace, oil ofbitter almonds, and cassia oil. Also useful flavorings are artificial,natural and synthetic fruit flavors such as vanilla, and citrus oilsincluding, without limitation, lemon, orange, lime, grapefruit, andfruit essences including apple, pear, peach, grape, strawberry,raspberry, cherry, plum, pineapple, apricot and so forth. Theseflavoring agents may be used in liquid or solid form and may be usedindividually or in admixture. Commonly used flavors include mints suchas peppermint, menthol, artificial vanilla, cinnamon derivatives, andvarious fruit flavors, whether employed individually or in admixture.Other useful flavorings include aldehydes and esters such as cinnamylacetate, cinnamaldehyde, citral diethylacetal, dihydrocarvyl acetate,eugenyl formate, p-methylamisol, and so forth may be used. In a specificaspect, the flavoring is spearmint oil. The flavor is optionally presentfrom about 0.1% to about 5% by weight of the antiviral composition.

Tablets may be molded tablets or compressed tablets. Tablets may beformed by wet granulation, dry granulation, and direct compression.These techniques are known to one of skilled in the art and aredescribed, for example, in the United States Pharmacopeia NationalFormulary USP XXII, 1990, pp. 1696-1697. Various other vitamins may beadded to composition. Tablets may optionally further comprise flavoringsor sweeteners. In one aspect, the sweetened, flavored tablet is utilizedas a lozenge to be dissolved in the mouth. The compositions of thedisclosure can also be prepared in a chewable form or an effervescentform. For effervescent preparations, the manufacturing method in thedisclosure is basically same as in the manufacturing method of the usualeffervescent preparations such as effervescent tablets. That is,components are weighed, mixed, and prepared directly by the powdercompression method, dry or wet granular compression method, etc. Orallydisintegrable tablets are described, for example, in U.S. Pat. No.7,431,942, Shimuzu et al., which is incorporated herein by reference.Lozenges with a hard candy base can be prepared, for example, by thetechniques of U.S. Pat. No. 6,316,008, Godfrey, which is incorporatedherein by reference.

A liquid composition may further comprise other nutrients. Such liquidcompositions may be prepared as described in U.S. Pat. No. 6,037,375,Sakamoto et al., which is incorporated herein by reference. A nutrientliquid composition of the disclosure contains a lysine, an ascorbiccompound, a flavonoid glycoside, a threonine and a pyridoxine asessential ingredients, and is prepared in the same manner as theordinary food and beverage, and other food materials may beappropriately added. As particularly preferred food materials,sweeteners such as organic acids and carbohydrates may be used. Organicacid components include citric acid, tartaric acid, malic acid, andsuccinic acid, and citric acid is particularly preferable. These organicacids are added usually in a range of 100 to 1500 mg/100 ml, preferably250 to 800 mg/100 ml, and the composition of the material in beverageform can be prepared.

Various sweeteners can be optionally used in the tablet, liquid,capsule, lozenge or troche formulations of the disclosure. Examples ofcarbohydrates and sweeteners include monosaccharides such as glucose andfructose, disaccharides such as maltose, sucrose, other ordinary sugars,sugar alcohols such as xylitol, sorbitol, glycerin and erythritol,polysaccharides such as dextrin and cyclodextrin, and oligosaccharidessuch as fructo-oligosaccharide, galacto-oligosaccharide andlacto-sucrose. Of the carbohydrates, as the components not adverselyaffecting the lipid metabolism, fructose and glycerin are preferred. Asoligosaccharide, addition of lacto-sucrose is preferred. A beveragecomposition of the disclosure can increase bifidobacteria in the body orlower the putrefaction products depending on the blend of thelacto-sucrose, so that the immune system can be intensified further.Other sweeteners include natural sweeteners such as thaumatin, steviaextract, rebaudioside A, glycyrrhizinic acid, etc. and syntheticsweeteners such as saccharin, aspartame, etc. These carbohydrates may bealso added as carbohydrate mixture such as isomerized sugar and refinedsugar. The sweetener is optionally present from about 0.1% to about 5%by weight of the solid composition. The blending of the carbohydratesmay be about 1 to 15 g in 100 ml of the beverage composition of thedisclosure, preferably about 3 to 12 g. The content of theoligosaccharide is about 0.5 to 10 g, preferably 1 to 3 g.

The nutrient liquid composition of the disclosure may also comprise,aside from the above, various nutrients, vitamins, minerals(electrolytes) including trace elements, perfumes including syntheticperfumes and natural perfumes, coloring matter, flavors (fruit, vanilla,chocolate, etc.), pectic acid and its salts, alginic acid and its salts,organic acids, thickener as protective colloidal substance, pHregulator, stabilizer, preservative, glycerins, alcohols, and sparklingcomponent for carbonated beverages. In addition, the composition of thedisclosure may also contain natural juice or fruit to be presented asfruit drink or vegetable drink. These may be used either alone or incombination of two or more kinds. The blending rate of these additivesis not particularly limited, and is generally selected in a range ofabout 0 to 20 parts by weight to 100 parts by weight of the compositionof the disclosure.

Optional additional vitamins include, whether water-soluble orfat-soluble, thiamine, niacin, retinol palmitate, bisbentiamine,riboflavin, cyanocobalamin, cholecalciferol, nicotinic acid amide,calcium pantothenate, folic acid, biotin, and choline ditartate, andthose belonging to vitamin B group.

The liquid nutrient composition of the disclosure is prepared byblending these components, and the method of preparation is notparticularly limited, and all components may be blended simultaneously,but more preferably fat soluble components are preliminarily dissolvedin oil, and water-soluble components in water, then the solution isemulsified by using an emulsifier, so that the composition of thedisclosure may be prepared. More preferably, the oil solution is addedto water and a proper emulsifier to emulsify, and an aqueous solution isadded and blended to the obtained emulsion. The blending operation ofthe components may be executed under ordinary temperature, or preferablyexecuted by slight heating operation.

The emulsification can be executed by using a proper emulsifyingmachine, for example, homo-mixer or high pressure homogenizer, either bycomplete passing system or by circulation system. The emulsion afteremulsification is filtered by conventional process, and poured intoproper containers and sterilized, so that a desired beverage product isobtained. Sterilization may be effected by heating, aseptic filtering,etc.

To prepare the composition of the disclosure as a carbonated beverage,carbon dioxide may be injected into the emulsifier by conventionalprocess. Such beverage is preferred to be prepared in the osmoticpressure range of about 260 to 600 mOsm/kg.

The liquid composition of the disclosure may be also prepared in aneffervescent form. The effervescent form should contain, aside from theessential components of the disclosure, proper amounts of sodiumcarbonate and/or sodium hydrogen carbonate and neutralizing agent asfoaming components. The neutralizing agent used herein is an acidiccompound capable of generating carbon dioxide by neutralizing sodiumcarbonate or sodium hydrogen carbonate. Such compound includes, forexample, L-tartaric acid, citric acid, fumaric acid, ascorbic acid andother organic acid. Preferred ascorbic acid possesses both the action ofneutralizing agent and the action of antioxidant.

It has surprisingly been found that compositions of the presentdisclosure inhibit influenza A viral replication in MDCK cells in vitro.QuantiGene Plex 2.0 assay utilizing branched DNA (bDNA) signalamplification and multi-analyte profiling beads (xMAP®) technologies wasused to enable the detection and quantitation of influenza A mRNA. Inparticular, Formula V was found to decrease influenza A mRNA moreeffectively than other known formulations such as Formula A2. In anotherembodiment, the disclosure provides a method to decrease viral activityand/or to reduce viral replication comprising treating a virus-infectedcell with a composition comprising a lysine, an ascorbic compound, aflavonoid glycoside, a threonine and a pyridoxine, in an effectiveamount.

Neuraminidase (also known as sialidase) is a very common enzyme thathydrolyzes terminal sialic acid residues on polysaccharide chains, mostoften exposing a galactose residue. Although neuraminidase is found inmammals, it is predominantly expressed in microorganisms such asbacteria and viruses. J. Biochem. Biophys. Methods 22, 23 (1991). Thenegative-stranded RNA influenza virus contains two surfaceglycoproteins, hemagglutinin (HA) and neuraminidase. Neuraminidase isthought to play a key role in the invasion of target cells and thesubsequent replication of the influenza virus through its cleavage oftarget cell receptor sialic acid moieties. This action prevents furtherinteraction of the virus with the target cell and facilitates elution ofprogeny virions from the infected cell (Haskell et al., Neuraminidaseinhibition and viral chemotherapy. J. Med. Chem. 13, 697 (1970);McKimm-Breschkin, Resistance of influenza viruses to neuraminidaseinhibitors—a review. Antiviral Res. 47, 1 (2000)). Additionally, newlysynthesized neuraminidase and HA on virions may also contain sialic acidresidues that can be cleaved by neuraminidase in order to preventself-aggregation. It is also thought that the penetration of mucosallinings by the virus is enhanced by neuraminidase hydrolytic action onfetuin, a major component of these membranes. These essential activitiesmake neuraminidase an important target for influenza drug development.Neuraminidase activity can be determined by use of a commerciallyavailable kit such as, for example, the Molecular Probes Amplex® RedNeuraminidase assay kit No. A22178 as used according to protocol, whichis incorporated herein by reference.

Yeo et al. investigated the effect of influenza A/Beijing/353/89 (H3N2)virus infection on the expression of type IV collagenase in twodifferent types of epithelial cells (Yeo et al. Influenza A virusinfection modulates the expression of type IV collagenase in epithelialcells. Arch. Virol. 144: 1361-1370 (1999)). Depending on the cell lineinfected, the viral infection caused changes in the expression of typeIV collagenase. The expression of matrix metalloproteinase-9 (MMP-9; 92kDa) but not of matrix metalloproteinase-2 (MMP-2; 72 kDa) wasstimulated in Vero cells. In MDCK cells, the MMP-2 production increasedwith the virus infection. The MMP-9 and -2 expression by influenza Avirus infection were determined to be modulated at transcriptionallevel, depending on the epithelial cell line. Therefore, formulationswhich inhibit expression of MMP-9 and/or MMP-2 may be of some interestas they may possess or enhance antiviral activity. It should be noted,however, that strong inhibition of these enzymes in pregnancy may not bedesirable.

In one embodiment, certain compositions of the disclosure were shown toinhibit neuraminidase in a dose-dependent fashion surprisingly betterthan known compositions. Results are shown in FIGS. 3-6. Formula V wasfound to inhibit neuraminidase more effectively than other knownformulations such as Formula A2.

In one embodiment, the disclosure provides a method to decrease viralactivity comprising treating a virus-infected cell with a compositionknown to inhibit neuraminidase activity and/or only weakly inhibit MMP-9expression, comprising a lysine, an ascorbic compound, a flavonoidglycoside, a threonine and a pyridoxine, in an effective amount. In oneaspect, a method for the treatment or prophylaxis of a viral infectionin a subject comprises administration to the subject a therapeuticallyeffective amount of a composition comprising a lysine, an ascorbiccompound, a flavonoid glycoside, a threonine, and a pyridoxine. One keyadvantage of the compositions of the present disclosure is the abilityto decrease viral activity in a non-strain specific manner.

Examples Example 1 Virus Production Protocol

Madin-Darby canine kidney (MDCK) cells (ATCC) were maintained in minimumessential medium (MEM) with Earle's salts (Gibco BRL, Grand Island,N.Y.) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/mlstreptomycin and 10% heat inactivated fetal calf serum, (Hyclone)) andbuffered by the addition of HEPES buffer solution (pH 7.55, 10 mM finalconcentration, Invitrogen).

The African Green Monkey kidney cell line Vero (ATCC) was maintained inminimum essential medium (MEM) with Earle's salts (Invitrogen)supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/mlstreptomycin and 10% heat inactivated fetal calf serum, (Hyclone).

The H1N1 influenza strain A/WS/33 (ATCC) was used to generate high titerviral stocks by passage in MDCK cells.

Example 2 In Vitro bDNA Assay for Influenza A mRNA Activity

MDCK or Vero cells were plated at 1e⁵ cells/mL in a 100 uL volume intissue culture treated 96 well plates. The following day cells wereinfected with virus at a multiplicity of infection of 10 and viralactivity was measured. For testing compounds, cells were preincubatedwith compound 1 hr prior to infection.

All formulations were run in triplicate for each concentration. Testformulations are shown in Example 5, Table 1. It should be noted thatFormula A1 is Airborne® effervescent health formula and Formula T isTamiflu®.

After overnight incubation, the cells were harvested, lysed, andsupernatants were subjected to QuantiGene Plex 2.0 assay (Panomics,Inc., Fremont, Calif.) for quantitative assay of influenza A mRNAaccording to manufacturer's protocol. The QuantiGene Plex 2.0 assaycombines branched DNA (bDNA) signal amplification and multi-analyteprofiling beads (xMAP®) technologies to enable the detection andquantitation of multiple mRNA targets simultaneously. Generally, thisassay is considered faster than traditional methods used to monitorinfluenza infection which typically require 2-5 days to perform.

The bDNA assay is a hybridization-based method of target-specific RNAquantitation that amplifies signal rather than target RNA, using labeledDNA probes. The QuantiGene Plex 2.0 system utilizes fluorescentmicrospheres (Capture Beads) as a support to capture specific RNAmolecules. The ability to quantify multiple target-specific RNAmolecules in a single sample lies in the design of the Probe Sets. Foreach RNA molecule of interest, an oligonucleotide Probe Set containingthree types of synthetic probes, Capture Extenders (CEs), LabelExtenders (LEs), and Blockers (BLs) that hybridize and span contiguoussequences of the target RNA, is provided. The CEs discriminate among thedifferent Capture Beads within the bead array while capturing, viacooperative hybridization, the target RNA.

Signal amplification is mediated by DNA amplification molecules thathybridize to the tails of the LEs. Each amplification unit containsmultiple hybridization sites for biotinylated Label Probes that bindStreptavidin-conjugated R-Phycoerythrin (SAPE). The resultingfluorescence signal associated with individual Capture Beads is read ona Luminex flow cytometer. Signal is reported as median fluorescenceintensity (MFI) and is proportional to the number of target RNAmolecules present in the untreated sample.

Results for influenza A-infected Vero cell supernatants are shown inFIGS. 1 and 2. In FIG. 1, Formula V and Formula 5 decreased the amountof influenza A mRNA in a dose dependent manner. Although Formula A1decreased the amount of amount of influenza A mRNA at high doses, it isinterpreted that the effervescent chemicals used to buffer the FormulaA1 commercial product increased the pH resulting in suppression by pHeffect at the higher concentrations of Formula A1. A1 was also found tobe cytotoxic at the high doses. Formula V appeared more potent thanFormula A2 in this assay as shown in FIG. 2. Concentrations areexpressed in micromolar (.1M) concentrations.

Example 3 In Vitro Molecular Probes Amplex® Red Neuraminidase(Sialidase) Assay

The Molecular Probes Amplex® Red Neuraminidase assay kit No. A22178 wasused according to manufacturer's protocol, which is incorporated hereinby reference.

The Molecular Probes Amplex® Red Neuraminidase assay utilizes Amplex Redto detect H₂O₂ generated by galactose oxidase oxidation of desialiatedgalactose, the end result of neuraminidase action. The H₂O₂ then, in thepresence of horseradish peroxidase (HRP), reacts with a 1:1stoichiometry with Amplex Red reagent to generate the red-fluorescentoxidation product, resorufin. (Mohanty et al. A highly sensitivefluorescent micro-assay of H₂O₂ release from activated human leukocytesusing a dihydroxyphenoxazine derivative. J. Immunol. Methods 202, 133(1997)). Resorufin has absorption and fluorescence emission maxima ofapproximately 571 nm and 585 nm, respectively, and because theextinction coefficient is high (54,000 cm⁻¹M⁻¹), the assay can beperformed either fluorometrically or spectrophotometrically.

MDCK or Vero cells were plated at 1e⁵ cells/mL in a 100 uL volume intissue culture treated 96 well plates. The following day cells wereinfected with virus at a multiplicity of infection of 10 and viralactivity was measured. For testing compounds, cells were preincubatedwith compound 1 hr prior to infection. After overnight incubation, thecells were harvested, lysed, and supernatants were subjected to assay.

All formulations were run in triplicate for each concentration. Testformulations are shown in Table 1. It should be noted that Formula A1 isAirborne® effervescent health formula and Formula T is Tamiflu®.

Results for influenza A-infected Vero cell supernatants are shown inFIGS. 3 and 4. Formula V is shown to be effective at inhibition of theneuraminidase enzyme in a dose-dependent manner. Although Formula A1seemed to be active at high doses, it is likely that the effervescentchemicals used to buffer the commercial product (Airborne®) increasedthe pH in the assay and resulted in suppression via a pH effect only atthe higher concentrations of Formula A1. Formula A1 also exhibitedcytotoxicity at high doses.

Example 4 In Vitro Inhibition of Matrix Metalloproteinase 9 (MMP-9)

A Quantikine Human MMP-9 (total) Immunoassay Assay, R&D Systems, wasused to quantify expression of MMP-9 in infected cells according to themanufacturer's protocol, which is incorporated herein by reference.

Vero cells were plated at 1e⁵ cells/mL in a 100 uL volume in tissueculture treated 96 well plates. The following day cells were infectedwith virus at a multiplicity of infection of 10 and viral activity wasmeasured. For testing compounds, cells were preincubated with compound 1hr prior to infection. After overnight incubation, the cells wereharvested, lysed, and supernatants were subjected to assay.

This MMP-9 assay employs the quantitative sandwich enzyme immunoassaytechnique. A monoclonal antibody specific for MMP-9 has been pre-coatedonto a microplate. Standards and samples are pipetted into the wells,and MMP-9 is bound by the immobilized antibody. After washing awayunbound substances, an enzyme-linked polyclonal antibody specific forMMP-9 is added to the wells. Following a wash to remove unboundantibody-enzyme reagent, a substrate solution is added to the wells andcolor develops in proportion to the amount of total MMP-9 (pro and/oractive) bound in the initial step. The color development is stopped andthe intensity of the color is measured.

Test formulations are shown in Example 5, Table 1. It should be notedthat Formula A1 is Airborne® effervescent health formula and Formula Tis Tamiflu®. Results for influenza A-infected Vero cell supernatants areshown in FIGS. 5 and 6. It is interpreted that the effervescentchemicals used to buffer the Formula A1 commercial product increased thepH in the assay and interfered with the assay via a pH effect only atthe higher concentrations of Formula A1.

Formula V weakly inhibited expression of MMP-9 in a dose-dependentmanner. Formula T (Tamiflu®) exhibited pronounced dose-dependentinhibition of expression of MMP-9. It is believed that the distinctlyweaker inhibition of MMP-9 expression exhibited in the presence ofFormula V may result in a decreased risk during pregnancy compared totreatment of a viral infection with Tamiflu® in a pregnant woman.

Example 5 In Vitro Inhibition of Matrix Metalloproteinase 2 (MMP-2)

Two separate kits were employed to determine if the formulations of thedisclosure inhibit expression of MMP-2. The Calbiochem® MMP-2 ELISA kitis a non-isotopic immunoassay for the in vitro quantification of humanMMP-2 protein in tissue culture medium. The R&D Systems Quantikine®MMP-2 (total) Immunoassay kit is used for the quantitative determinationof active and pro-Matrix Metalloproteinase 2 (total MMP-2)concentrations in cell culture supernates. All formulations were run intriplicate utilizing each of the two assay kits. No formulation of thepresent disclosure significantly inhibited MMP-2 expression when testedin various concentrations up to about 5 μM.

Example 6 Formulations. Powder Formulations

Ingredients for various test formulations are shown in Table 1; theamounts for a single dose packet of powder are shown. A test batch foreach formulation was prepared utilizing five times the amounts shown.Each ingredient was added to the batch and the batch was mixedthoroughly, divided by weight and sealed into five packets.Alternatively, the powder can be filled to hard shell capsules.

TABLE 1 Formulations- weight per dose for powder formulation. Amount(grams) Formula Formula Formula Formula Formula Material V F 5 K A2L-Lysine HCl 3.00 3.00 3.00 3.00 1.00 Proline 0.75 Arginine 0.50Threonine 0.05 0.06 0.05 0.05 Acetyl-L-cysteine 0.20 0.20Selenomethionine 0.01 Ascorbic acid 0.28 1.00 0.45 0.20 0.71 Calciumascorbate 0.64 0.64 0.64 0.02 Magnesium ascorbate 0.05 Niacinamideascorbate 0.30 0.42 Ascorbyl palmitate 0.05 0.05 0.05 Green tea extract(90% 1.0 polyphenols) Hesperidin complex 0.30 0.33 0.30 0.30 Rutin NF0.30 0.33 0.30 0.30 Pyridoxine HCl 0.05 0.05 0.05 0.05 Taurine 0.20 0.040.20 Copper gluconate 0.02 Manganese gluconate 0.01 dihydrate Pectin(87% 0.33 galacturonic acid) Total weight per 5.17 5.14 5.03 5.20 4.23packet (g)

Example 7 Formulations. Tablet Formulations

Tablet formulations were prepared using the ingredients shown in Table2. The amount of ingredient shown is equal to one dose. Five tabletscontained one dose of the formulation. A wet granulation technique wasemployed and compressed tablets were coated with a white coating shownin Table 2. Tablets were dried and stored at room temperature.

TABLE 2 Tablet Formulations- amount per tablet. Formula FormulaIngredient FT (mgs) VT (mgs) Citrus Bioflavonoid (Hesperidin) 142.9766.60 98% Methocel E-5 HPMC 4.42 5.00 L-Lysine Monohydrate 612.20 600.00Ascorbic Acid 218.80 200.00 Rutin NF 30.00 66.00 Pectin 30.00 66.00Pyridoxine HCl USP Vit B6 13.40 10.00 Taurine 8.20 8.00 L-Threonine12.20 12.00 Microcrystalline Cellulose 200.00 200.00 CroscarmelloseSodium 20.00 20.00 Silica 6.00 6.00 Magnesium Stearate 10.00 10.00Riboflavin Vit B2 1.50 0 Purified Water White Coating including: 40.0040.00 Dextrin Dextrose Hypermellos Mineral Oil Polyethylene GlycolPolysorbate 80 Croscarmellose Sodium Sodium Citrate Titanium DioxideTotal Mgs: 1349.69 1308.82

Example 8 Preliminary Clinical Data

A preliminary trial was performed during an influenza A outbreak.Subjects were given the tablet Formulation FT of Example 6, wherein 5tablets equaled one dose. Study subjects were instructed to take onedose at the first sign of flu-like symptoms; and repeat dosing every 4to 6 hours until symptoms resolved. Subjects swallowed each dose withwater. Fourteen patient surveys were completed. Subjects reportedflu-like symptoms as shown in Table 3. In addition, certain patientsalso exhibited additional symptoms of dry cough, sore throat, orheadache.

TABLE 3 Preliminary Clinical Surveys. Duration Symptoms of PatientGender Body Symptoms No. Age F M Aches Fatigue Fever (hours) 1 42 1 0 11 0 1.5 2 21 0 1 0 0 1 2 3 49 0 1 1 1 1 1.5 4 85 0 1 1 0 1 2 5 27 0 1 11 1 2 6 26 1 0 1 1 1 1 7 17 1 0 0 1 1 2 8 54 1 0 0 1 0 1.5 9 49 1 0 1 11 3.5 10 52 1 0 1 1 1 4.5 11 22 1 0 1 1 1 5 12 43 0 1 1 1 1 12 13 61 0 11 1 1 13 14 54 1 0 1 1 1 18 Average 42 8 6 11 12 12 4.97 Std. Dev. 195.36

Eight subjects reported resolution of symptoms within about 2 hoursafter taking the first dose of the formula. Three more subjects reportedcomplete resolution of symptoms within about 6 hours after the first oftwo doses. Three more subjects reported resolution within about 18 hoursafter the first of three to four doses.

Example 9 Prophylactic Dosing

In an anecdotal study, two subjects working in a vision center on adaily basis were asked not to get a flu vaccine last season but to takea single daily prophylactic dose of Formula F at 40% strength (2 tabletsper dose). Each subject reported that a co-worker had contractedinfluenza during this time. The area in which the subjects live and workexperienced a major outbreak of influenza A, with two local hospitalsbanning visitors during the outbreak. Neither subject contractedinfluenza.

Example 10 Neuraminidase Assay of Formulation Components

In order to determine the relative contribution of each component in thecompositions of the disclosure and identify additive and synergisticeffects, each component of interest was evaluated in variouscombinations in the Neuraminidase Assay in a manner similar to Example3. The test compositions were assayed in an in vitro assay to measureneuraminidase activity on influenza A infected MDCK cells. Madin DarbyCanine Kidney (MDCK) epithelial cells were obtained from the ATCC(Manassas, Va.) and grown as described by the vendor. Cells wereinfected with human influenza virus (TC-adapted strain A/WS/33,ATCC-1520) and propagated as described previously. This straindemonstrates cross reaction with PR8 and swine influenza virus accordingto the ATCC. To measure neuraminidase activity, viral stocks were usedto infect MDCK cells and neuraminidase activity was quantified using theAmplex Red Neuraminidase assay kit (Invitrogen, Inc. Carlsbad Calif.).Briefly, MDCK cells were infected with influenza A virus at 37° C. inthe presence of the compositions shown in Table 4. Each condition wasassayed in triplicate over an 8 point 2 fold dilution series. The AmplexRed method of quantifying neuraminidase activity uses three differentenzymes: NA, galactose oxidase and horseradish peroxidase. The combinedaction of peroxide and HRP, Amplex Red is oxidized to liberate resorufinand read on a spectrophotometer at OD 563 nm.

Oseltamivir carboxylate was used as a standard neuraminidase inhibitor(NAI) positive control for comparison. Tamiflu® (Oseltamivir phosphate)is an ethyl ester prodrug requiring ester hydrolysis for conversion tothe active form, oseltamivir carboxylate. Oseltamir carboxylate is aninhibitor of influenza virus neuraminidase affecting release of virusparticles.

FIG. 7, Table 4, shows the various compositions that were tested in theneuraminidase assay. Each test composition was compared to Formula V andto oseltamivir carboxylate in the neuraminidase assay. FIGS. 8-13 showthe activity of various compositions in the neuraminidase assay. Theconcentration shown in the x-axis of the FIGS. 8-13 represents eitherthe L-lysine HCl concentration in each test composition and in FormulaV, or the oseltamivir carboxylate concentration.

FIG. 8 shows an overlay of the neuraminidase activity of human influenzavirus infected cells after exposure to oseltamivir carboxylate, FormulaV and ten test compositions containing various components of Formula V.The numbers 1-6 are used to identify individual major components ofFormula V. All six components, lysine, ascorbic acid/ascorbates,flavonoid glycosides, pyridoxine, threonine and taurine are necessaryfor full synergistic inhibition of neuraminidase activity.

FIG. 9 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine alone (1) or lysine with ascorbic acid/ascorbates (1/2; 9A).Neither test composition is very effective until the highestconcentration of lysine/ascorbates (1/2; 9B) is employed.

FIG. 10 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/flavonoid glycosides (1/2/3; 10A) orlysine/ascorbates/pyridoxine (1/2/4; 10B). Some dose-dependent decreasein neuraminidase activity is seen with the formulation oflysine/ascorbates/flavonoid glycosides (1/2/3) at the four highestconcentrations. A weaker dose-dependent decrease in neuraminidaseactivity is seen with the formulation of lysine/ascorbates/pyridoxine(1/2/4) at the four highest concentrations.

FIG. 11 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/threonine (1/2/5; 11A) or lysine/ascorbates/taurine(1/2/6; 11B). A weak decrease in neuraminidase activity is seen withlysine/ascorbates/threonine at higher concentrations.

FIG. 12 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/pyridoxine HCl/threonine (1/2/4/5; 12A) orlysine/ascorbates/flavonoid glycosides/threonine (1/2/3/5; 12B). Thecomposition of lysine/ascorbates/flavonoid glycosides/threonine exhibitsa dose dependent decrease in neuraminidase activity at the three highestconcentrations.

FIG. 13 shows neuraminidase activity of human influenza virus infectedcells after exposure to oseltamivir carboxylate, Formula V and eitherlysine/ascorbates/flavonoid glycosides/pyridoxine HCl/threonine(1/2/3/4/5; 13A) or lysine/ascorbic acid/flavonoid glycosides/pyridoxineHCl/threonine/taurine (1/2/3/4/5/6; 13B). Both compositions produce arobust, dose dependent decrease in neuraminidase activity. However, onlythe composition of lysine/ascorbic acid/flavonoid glycosides/pyridoxineHCl/threonine/taurine is comparable to Formula V. Formula V and1/2/3/4/5/6 differ by the type of ascorbic compound utilized. Formula Vcomprises ascorbic acid/mixed ascorbates as shown in Table 1. Thecomposition 1/2/3/4/5/6 utilized in FIG. 13B comprises only ascorbicacid and not the mixed ascorbates as shown in Table 4.

Each of the six components, 1/2/3/4/5/6, appears to be required for thefull desired dose-dependent suppression of neuraminidase activity inthis assay. The composition of lysine/ascorbates/flavonoidglycosides/pyridoxine HCl/threonine (1/2/3/4/5) exhibited dose dependentsuppression across the full range of concentrations tested. Thecompositions 1/2/3; 1/2/4; and 1/2/3/5 each exhibited dose dependentsuppression of neuraminidase activity in this assay only at the higherconcentrations tested.

We claim:
 1. An antiviral supplement oral dosage form comprising acomposition comprising a therapeutically effective amount of a lysine ina single dose range of from about 1,000 mg to about 5,000 mg, anascorbic compound, a flavonoid glycoside, a threonine, a taurine and apyridoxine.
 2. The dosage form of claim 1, wherein the dosage form is ina form selected from the group consisting of powder, capsule, lozenge,troche, tablet, liquid, and caplet.
 3. The dosage form of claim 1,wherein the dosage form is a solid dosage form.
 4. The dosage form ofclaim 1, wherein the dosage form is a chewable form.
 5. The dosage formof claim 1, wherein the dosage form is a divided dosage form.
 6. Thedosage form of claim 1, wherein the lysine is selected from L-lysine,L-lysine monohydrochloride, L-lysine dihydrochloride, L-lysinesuccinate, L-lysine glutamate, and L-lysine orotate.
 7. The dosage formof claim 1, wherein the ascorbic compound is selected from one or moreof ascorbic acid, calcium ascorbate, magnesium ascorbate, potassiumascorbate, sodium ascorbate, manganese ascorbate, zinc ascorbate, ironascorbate, copper ascorbate, boron ascorbate, molybdenum ascorbate,chromium ascorbate, ascorbyl palmitate, ascorbyl arachidonate, ascorbylstearate, ascorbyllinoleate, ascorbyl linoleneate, and ascorbyl oleate.8. The dosage form of claim 1, wherein the flavonoid glycoside isselected from the group consisting of hesperidin and rutin, naringin,and quercitrin.
 9. The dosage form of claim 1, wherein the flavonoidglycoside comprises hesperidin and rutin.
 10. The dosage form of claim1, wherein the flavonoid glycoside comprises hesperidin and rutin in a1:1 weight ratio.
 11. The dosage form of claim 1, wherein the pyridoxineis pyridoxine hydrochloride.
 12. The dosage form of claim 1, wherein thecomposition comprises L-lysine monohydrochloride, ascorbic acid,hesperidin, rutin, threonine, taurine, and pyridoxine hydrochloride. 13.The dosage form of claim 12, wherein the composition further comprisingan additional ascorbic compound selected from the group consisting ofcalcium ascorbate, niacinamide ascorbate, and ascorbyl palmitate. 14.The dosage form of claim 1, wherein the dosage form further comprises anadditive selected from the group consisting of vehicle, binder,disintegrating agent, lubricant, thickener, surfactant, osmotic pressureregulator, electrolyte, sweetener, flavoring, perfume, pigment, and pHregulator.
 15. The dosage form of claim 13, wherein the disintegratingagent is selected from the group consisting of microcrystallinecellulose, carboxymethylcellulose (CMC), CMC-Na, CMC-Ca, andcroscarmellose sodium.
 16. The dosage form of claim 14, wherein thelubricant is selected from the group consisting of leucine, isoleucine,valine, sugar-ester, hardening oil, stearic acid, magnesium stearate,talc, and macrogol.
 17. The dosage form of claim 14, wherein thesweetener is selected from the group consisting of glucose, fructose,maltose, sucrose, xylose, lactose, xylitol, sorbitol, mannitol,maltitol, xylitol, coupling sugar, paratinose, glycerin, erythritol,dextrin, cyclodextrin, fructo-oligosaccharide, galacto-oligosaccharide,lacto-sucrose, thaumatin, stevia, stevia extract, rebaudioside A,glycyrrhizinic acid, saccharin, alitame, and aspartame.
 18. The dosageform of claim 1, wherein a single dose comprises from about 2 g to about3.5 g L-lysine monohydrochloride; from about 0.1 g to about 1.5 gascorbic acid; from about 0.2 g to about 0.8 g hesperidin; from about0.1 g to about 0.5 g rutin; from about 0.04 g to about 0.08 g pyridoxinehydrochloride; from about 0.01 g to about 0.08 g threonine; and fromabout 0.02 g to about 0.4 g taurine.
 19. The dosage form of claim 18,further comprising from about 0.5 g to about 0.75 g calcium ascorbate;from about 0.1 g to about 0.5 g niacinamide ascorbate; and from about0.01 g to about 0.1 g ascorbyl palmitate.
 20. A method of treating orpreventing a viral infection in a subject in need thereof comprisingadministering to the subject a dosage form comprising a therapeuticallyeffective amount of a composition comprising a lysine in a single doserange of from about 1,000 mg to about 5,000 mg, an ascorbic compound, aflavonoid glycoside, a threonine, a taurine and a pyridoxine.
 21. Themethod of claim 20, wherein the viral infection is selected from thegroup consisting of influenza A, influenza B, influenza C and non-polioenterovirus infection.
 22. The method of claim 20, wherein the dosageform is selected from the group consisting of powder, capsule, lozenge,troche, tablet, liquid, or caplet.
 23. The method of claim 20, whereinthe dosage form is a solid dosage form.
 24. The method of claim 20,wherein the dosage form is a chewable form.
 25. The method of claim 20,wherein the lysine is selected from L-lysine, L-lysinemonohydrochloride, L-lysine dihydrochloride, L-lysine succinate,L-lysine glutamate, and L-lysine orotate.
 26. The method of claim 20,wherein the ascorbic compound is selected from one or more of ascorbicacid, calcium ascorbate, magnesium ascorbate, potassium ascorbate,sodium ascorbate, manganese ascorbate, zinc ascorbate, iron ascorbate,copper ascorbate, boron ascorbate, molybdenum ascorbate, chromiumascorbate, ascorbyl palmitate, ascorbyl arachidonate, ascorbyl stearate,ascorbyl linoleate, ascorbyl linoleneate, and ascorbyl oleate.
 27. Themethod of claim 20, wherein the flavonoid glycoside comprises hesperidinand rutin.
 28. The method of claim 20, wherein the composition comprisesL-lysine monohydrochloride, ascorbic acid, calcium ascorbate,niacinamide ascorbate, ascorbyl palmitate, hesperidin, rutin, pyridoxinehydrochloride, threonine, and taurine.
 29. The method of claim 28,wherein the composition further comprises calcium ascorbate, niacinamideascorbate, and ascorbyl palmitate.
 30. A method to decrease the severityor duration of symptoms of a viral infection in a subject in needthereof comprising administering to the subject a dosage form comprisinga therapeutically effective amount of a composition comprising a lysinein a single dose range of from about 1,000 mg to about 5,000 mg, anascorbic compound, a flavonoid glycoside, a threonine, a taurine and apyridoxine.
 31. The method of claim 30, wherein the viral infection isselected from the group consisting of influenza A, influenza B,influenza C and non-polio enterovirus infection.
 32. The method of claim30, wherein the dosage form is selected from the group consisting ofpowder, capsule, lozenge, troche, tablet, liquid, or caplet.
 33. Themethod of claim 30, wherein the dosage form is a solid dosage form. 34.The method of claim 30, wherein the dosage form is a chewable form. 35.The method of claim 30, wherein the lysine is selected from L-lysine,L-lysine monohydrochloride, L-lysine dihydrochloride, L-lysinesuccinate, L-lysine glutamate, and L-lysine orotate.
 36. The method ofclaim 30, wherein the ascorbic compound is selected from one or more ofascorbic acid, calcium ascorbate, magnesium ascorbate, potassiumascorbate, sodium ascorbate, manganese ascorbate, zinc ascorbate, ironascorbate, copper ascorbate, boron ascorbate, molybdenum ascorbate,chromium ascorbate, ascorbyl palmitate, ascorbyl arachidonate, ascorbylstearate, ascorbyl linoleate, ascorbyl linoleneate, and ascorbyl oleate.37. The method of claim 30, wherein the flavonoid glycoside compriseshesperidin and rutin.
 38. The method of claim 30, wherein thecomposition comprises L-lysine monohydrochloride, ascorbic acid, calciumascorbate, niacinamide ascorbate, ascorbyl palmitate, hesperidin, rutin,pyridoxine hydrochloride, threonine, and taurine.
 39. The method ofclaim 38, wherein the composition further comprises calcium ascorbate,niacinamide ascorbate, and ascorbyl palmitate.
 40. The method of claim30, wherein the symptoms are selected from the group consisting of bodyaches, fatigue, fever, dry cough, sore throat, and headache.
 41. Themethod of claim 40, wherein the administering is performed at the firstsign of flu-like illness indicated by one or more of the symptoms, atintervals selected from the group consisting of every four waking hours,every six waking hours, and twice a day, until symptoms are resolved.